1. Field of the Invention
A method is provided for simultaneously detecting lipids and phosphatidate intermediates in cells incubated with stable-isotope labeled fatty acids. The method may be used for screening compounds that modulate triglyceride biosynthesis in a high-throughput format.
2. Description of the Related Art
Triglyceride or triacylglycerol is a major transport source and energy storage in eukaryotes. Triglyceride is synthesized from a glycerol molecule and three fatty acid molecules. Each of the fatty acid molecules is attached, via an ester bond, to each of three hydroxyl groups of the glycerol molecule. Triglyceride, as many neutral lipids, contains fatty acid molecules in various chain lengths with a common length of 16, 18, or 20 carbons.
The two major biosynthetic pathways of triglyceride are the glycerol-3-phosphate pathway, which exists predominately in liver and adipose tissue, and a monoacylglycerol pathway, which exists predominately in the intestine. The glycerol-3-phosphate pathway, generating more than 90% of triglyceride in liver, is illustrated below:
Wherein FA-CoA is fatty acid CoA, GPAT is glycerol-3-phosphate acyltransferase, AGPAT is 1-acylglycerol-3-phosphate-O-acyltransferase, PAP is phosphatidic acid phosphatase and DGAT is acyl-coA: diacylglycerol acyltransferase.
The final step of the glycerol-3-phosphate biosynthetic pathway can be catalyzed by either DGAT1 or DGAT2 (Cases et al. 1998, Proc Natl Acad Sci USA 95:13018; Cases et al. 2001 J Biol Chem 276:38870). Although DGAT1 and DGAT2 both convert diglyceride to triglyceride, they do not share similarity in either nucleotide or amino acid sequences. It has been reported that knockout mice lacking DGAT1 (Dgat1-/-) do not display obvious changes in triglyceride metabolism in the liver (Smith et al. 2000, Nat Genet. 25:87). In addition, knockout mice lacking DGAT2 (Dgat2-/-) display severely reduced triglyceride content in the liver (Stone et al. 2004, J Biol Chem 279:11767). Further, studies have shown that suppression of DGAT2 with antisense oligonucleotides reduces hepatic triglyceride content in rodents (Chol et al. 2007, J Biol Chem 282:22678; Liu et al. 2008, Biochim Biophy Acta 1781:97) and reverses diet-induced hepatic steatosis and insulin resistance in rats (Chol et al. 2007, J Biol Chem 282:22678). These studies suggest that DGAT1 and DGAT2 function differently in triglyceride biosynthesis. Therefore, specific targeting of either DGAT1 or DGAT2 may provide benefit in modulating triglyceride with limited toxicity.
Disorder or imbalance in triglyceride metabolism has been associated with pathogenesis and increased risk for obesity, metabolic syndrome, type II diabetes, nonalcoholic fatty liver disease and coronary heart disease (Lewis et al. 2002, Endocrine Reviews 23:201; Malloy and Kane 2001, Adv Intern Med 47:111). Therefore, compounds that modulate enzymatic activity within the triglyceride biosynthetic pathway, including DGAT1 and DGAT2, may be useful as potential therapeutic targets for metabolic diseases.
The radioactive substrates and a thin-layer chromatography have been widely used to monitor triglyceride synthesis (Stone et al. 2004, J Biol Chem 279:11767; Zhu et al. 2002, Atherosclerosis 164:221). Stone et al. labels triglycerides with 3H-glycerol in primary hepatocytes and detects the radioisotope-labeled triglycerides using thin-layer chromatography and radio image analysis (Stone et al. 2004, J Biol Chem 279:11767). Similarly, Zhu et al. labels triglycerides with 3H-oleic acid in human hepatocarcinoma cells and detects the labeled triglycerides using thin-layer chromatography (Zhu et al. 2002, Atherosclerosis 164:221).
Recently, Magkos et al. uses the mass spectrometry technology to detect neutral lipids (Magkos et, al. 2007, J Lipid Research 48: 1204). Magkos et al. administers 1,1,2,3,3-2H-glycerol and 2,2-2H-palmitate in vivo, and extracts very low density lipoprotein triglycerides from plasma by chloroform/methanol and ultracentrifugation. Magkos et al. detects labeled glycerol and methylated palmitate released from triglycerides using a gas chromatography-mass spectroscopy system (Magkos et al. 2007, J Lipid Research 48:1204). Magkos et al. does not detect intact triglyceride, i.e. specific triglyceride molecules, nor different intermediates in the triglyceride pathways.
As far as applicants know, the existing assays do not monitor or detect the intermediates including lysophosphatidic acid, phosphatidic acid and diacylglycerol generated during the triglyceride biosynthesis. Also, the existing assays require additional extraction procedures and labor-intensive detection techniques. Further, these methods have limited throughput and are difficult to be adapted for high-throughput format for screening large numbers of compounds. Thus, there is still a need to develop a method for analyzing triglyceride biosynthesis and identifying compounds, which modulate the triglyceride pathway in a high-throughput format.